• Sample, Clare (PI)
  • Sample, Jeffery (PI)

Project: Research project

Project Details


The long-term objective of the proposed research is to delineate the
mechanisms that differentially regulate Epstein-Barr virus (EBV) gene
expression in latently infected B lymphocytes. The potential of EBV to
cause or contribute to the majority of its associated disease states relies
on the maintenance of a latent infection. Recent data indicate that there
are two states of EBV latency, gpI and gpIII. Establishment of latency
depends on the actions of one or more of the nine viral proteins expressed
in the gpIII state of latency. Based on the more restricted EBV gene
expression in Burkitt lymphoma (gpI latency), we postulate viral gene
expression is subsequently, or intermittently, restricted to the nuclear
protein EBNA-1, necessary for replication of the viral genome, and to
possibly as yet unknown proteins. Restricted expression would enable the
production of progeny latently-infected cells and evasion of host immune
surveillance. We propose the following aims to reach our long-term
objectives: 1) Elucidate the mechanisms regulating transcription of the
EBNA genes during gpI and gpIII latency; 2) Characterize the regulation of
selective 3' processing and alternative splice-site selection in the
maturation of the EBNA mRNAs; 3) Identify and characterize novel EBV genes
expressed in gpI Burkitt lymphoma cells.

During gpIII latency the 6 EBNA proteins are encoded by mRNAs derived by
differential pre-mRNA processing of a common primary transcript. The
central mechanism permitting EBNA-1 expression in the absence of the other
EBNAs in gpI latency is a switch to a EBNA-1-specific promoter. By using
a short-term expression assay in which the different EBNA promoters are
linked to reporter genes, we will identify (under Aim 1) the mode of
regulation of differential transcription of the EBNA genes in gpI and gpIII
latently infected B cells. Subsequent characterization of positive and
negative regulatory elements within these promoters will permit us to
delineate the actual mechanisms regulating the shifts between gpI and gpIII
latency. In Aim 2 we will employ in vivo and in vitro RNA processing
assays to examine the regulatory roles of alternative splicing and 3'
processing in the differential expression of the EBNA mRNAs in gpIII
latency, and exon skipping in the generation of the gpI EBNA-1 mRNA.
Finally, through a process of characterization of mRNAs/cDNAs and
identification of the proteins they encode, we will determine whether there
are additional EBV genes expressed in gpI Burkitt cells that may be
critical to this disease state (Aim 3).
Effective start/end date4/1/9212/31/06